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Hopes, Amanda, Nekrasov, Vladimir, Kamoun, Sophien 
ORCID: https://orcid.org/0000-0002-0290-0315 and Mock, Thomas ORCID: https://orcid.org/0000-0001-9604-0362
(2016)
Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana.
Plant Methods, 12 (1).
ISSN 1746-4811
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Abstract
Background: CRISPR-Cas is a recent and powerful addition to the molecular toolbox which allows programmable genome editing. It has been used to modify genes in a wide variety of organisms, but only two alga to date. Here we present a methodology to edit the genome of Thalassiosira pseudonana, a model centric diatom with both ecological significance and high biotechnological potential, using CRISPR-Cas. Results: A single construct was assembled using Golden Gate cloning. Two sgRNAs were used to introduce a precise 37 nt deletion early in the coding region of the urease gene. A high percentage of bi-allelic mutations (≤61.5%) were observed in clones with the CRISPR-Cas construct. Growth of bi-allelic mutants in urea led to a significant reduction in growth rate and cell size compared to growth in nitrate. Conclusions: CRISPR-Cas can precisely and efficiently edit the genome of T. pseudonana. The use of Golden Gate cloning to assemble CRISPR-Cas constructs gives additional flexibility to the CRISPR-Cas method and facilitates modifications to target alternative genes or species.
| Item Type: | Article |
|---|---|
| Additional Information: | This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
| Uncontrolled Keywords: | crispr-cas,diatom,genome editing,urease,golden gate,thalassiosira pseudonana |
| Faculty \ School: | Faculty of Science > School of Environmental Sciences Faculty of Science Faculty of Science > The Sainsbury Laboratory Faculty of Science > School of Biological Sciences |
| UEA Research Groups: | Faculty of Science > Research Groups > Plant Sciences Faculty of Science > Research Centres > Centre for Ecology, Evolution and Conservation Faculty of Science > Research Groups > Environmental Biology Faculty of Science > Research Groups > Marine and Atmospheric Sciences (former - to 2017) Faculty of Science > Research Groups > Resources, Sustainability and Governance (former - to 2018) Faculty of Science > Research Groups > Centre for Ocean and Atmospheric Sciences |
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| Depositing User: | Pure Connector |
| Date Deposited: | 25 Nov 2016 16:17 |
| Last Modified: | 13 Dec 2024 01:23 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/61490 |
| DOI: | 10.1186/s13007-016-0148-0 |
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